应用髓源性抑制细胞来源外泌体治疗糖尿病肾病小鼠  

Study on the treatment of diabetic nephropathy mice with myeloid-derived suppressor cell-produced exosomes

作　　者：梁丽 张一珂 邵烁鋆 谢劲松[1] 朱栋炜 LIANG Li;ZHANG Yi-ke;SHAO Shuo-yun;XIE Jing-song;ZHU Dong-wei(Department of Clinic Laboratory,Nanjing Hospital Affiliated to Nanjing University of Traditional Chinese Medicine,Nanjing 210003,China;Department of Immunology,Jiangsu Key Laboratory of Laboratory Medicine,School of Medicine Jiangsu University,Zhenjiang 212013,China)

机构地区：[1]南京中医药大学附属南京医院(南京市第二医院)检验科,南京210003 [2]江苏大学医学院免疫学教研室,江苏省临床检验诊断重点实验室,镇江212013

出　　处：《现代免疫学》2024年第6期474-481,共8页Current Immunology

基　　金：国家自然科学基金青年基金项目(82202001);江苏省大学生创新创业计划(2022102991193X)。

摘　　要：已知糖尿病肾病(diabetic nephropathy,DN)与免疫应答的过度激活有关,而髓源性抑制细胞(myeloid-derived suppressor cell,MDSC)来源的外泌体(exosome,EX)展现出强大的免疫抑制能力,研究探讨MDSC-EX对DN模型小鼠病情的影响。经高脂饮食和链脲佐菌素(streptozotocin,STZ)注射构建DN小鼠模型,磁珠分选DN小鼠脾脏中MDSC的两个亚群——多形核样MDSC(polymorphonuclear MDSC,PMN-MDSC)和单核细胞样MDSC(monocytic MDSC,MO-MDSC)。培养PMN-MDSCs和MO-MDSCs并收集上清液,制备EX,FACS检测EX对CD3+T细胞增殖的抑制作用。尾静脉注射PMN-MDSC-EX或MO-MDSC-EX至DN模型小鼠,观察处理对模型小鼠空腹血糖(fasting blood glucose,FBG)、体质量(body weight,BW)、24 h尿液总蛋白定量(total protein in urine within 24 hours,TPU)、血清尿素氮(urea nitrogen in serum,UNS)、血清肌酐(creatinine in serum,CS)以及肾小球滤过率(glomerular filtration rate,GFR)的影响。显微镜下观察各组肾脏H-E染色情况,Western blotting检测肾脏组织纤维连接蛋白(fibronectin)、Ⅳ型胶原(collagen typeⅣ,collagenⅣ)和α平滑肌肌动蛋白(alpha smooth muscle actin,α-SMA)的表达情况。实验成功构建DN模型并制得PMN-MDSC-EX和MO-MDSC-EX。相较MO-MDSC-EX,PMN-MDSC-EX可更显著地抑制CD3+T细胞增殖(P<0.05)。相较MO-MDSC-EX,PMN-MDSC-EX显著降低DN小鼠FBG、TPU、UNS和CS水平,增加模型鼠BW和GFR(均P<0.05)。PMN-MDSC-EX和MO-MDSC-EX干预后DN小鼠肾脏组织损伤明显减轻,相较MO-MDSC-EX,PMN-MDSC-EX抑制DN模型小鼠肾小球硬化的作用更为显著(均P<0.05)。由此,相较MO-MDSC-EX,PMN-MDSC-EX具有更强的缓解DN小鼠病情的作用,提示PMN-MDSC-EX或可成为治疗DN的新途径。Myeloid-derived suppressor cells(MDSCs)are a heterogeneous population of bone marrow-derived cells that are abundantly accumulated in patients and mouse models of diabetic nephropathy(DN).Studies have shown that MDSC-derived exosomes(EXs)have obvious immuno-suppressive functions,which may inhibit the development of inflammation in DN.This study aims to investigate the immuno-suppressive capacity of EX derived from MDSC(MDSC-EX)and its effect on DN mice.DN mice were established through high-fat diet and intra-peritoneal injection of streptozotocin(STZ).Two subsets of MDSC,polymorphonuclear MDSC(PMN-MDSC)and monocytic MDSC(MO-MDSC),were isolated from DN mice spleens by magnetic-activated cell-sorting(MACS).PMN-MDSC and MO-MDSC-derived EXs were prepared from the culture supernatants of PMN-MDSCs and MO-MDSCs and FACS was used to examine their inhibitory effect on CD3+T cell proliferation.PMN-MDSC-EX or MO-MDSC-EX were caudal vein injected into DN mice and the effects on fasting blood glucose(FBG),body weight(BW),total protein in urine within 24 hours(TPU),urea nitrogen in serum(UNS),creatinine in serum(CS)and glomerular filtration rate(GFR)were observed.H-E staining of kidney was observed under microscope and the protein expressions of fibronectin,collagen typeⅣ(collagenⅣ),and alpha smooth muscle actin(α-SMA)in kidney tissues were measured by western blotting.The results showed the successful establishment of DN mouse model and the isolation of PMN-MDSC-EX and MO-MDSC-EX.Compared to MO-MDSC-EX,PMN-MDSC-EX had a greater capacity to inhibit the proliferation of CD3+T lymphocytes(P<0.05).Compared to MO-MDSC-EX,PMN-MDSC-EX decreased the FBG,TPU,UNS,and CS in DN mice to a larger extent,and increased the BW and GFR significantly(all with P<0.05).Intervention with both PMN-MDSC-EX and MO-MDSC-EX significantly alleviated kidney tissue damage in DN mice.Compared to MO-MDSC-EX,PMN-MDSC-EX presented a better inhibitory effect on glomerulosclerosis in model mice.In conclusion,PMN-MDSC-EX has a stronger protective effect on

关 键 词：髓源性抑制细胞 外泌体 糖尿病肾病 

分 类 号：R392.6[医药卫生—免疫学]